Like transcription factors, microRNAs (miRNAs), a class of short regulatory non-coding RNAs known for their role in organ development, cellular differentiation, homeostasis, and function, have been extensively studied for their roles in controlling expression of different sets of genes that dictates the outcome of developmental transitions or cellular activation status of the immune cell populations. Previously, we have identified an important miRNA family, miR-23~27~24 clusters that play a diverse role in regulating the differentiation and function of multiple CD4+ helper T (Th) cell lineages as well as regulatory T (Treg) cells. Our work has shown that proper gene regulation by the miR-23~27~24 in CD4+ T cells is crucial to ensure the optimal balance between immunity and tolerance. Loss of miR-23~27~24 clusters in T cells resulted in dysregulated follicular helper (Tfh) cell responses when mice aged and severe Th2-driven airway inflammation upon challenges of different allergens. On the other hand, excessive expression of members of this miRNA family would also lead to the development of autoimmunity through both promoting the proinflammatory cytokine production by effector T (Teff) cells as well as impairing Treg cell homeostasis and function. Considering that many of the miRNA family targets identified in our previous work are also known to function in other immune cell populations, miR-23~27~24 clusters likely have a broader impact on the immune system beyond their role in regulating CD4+ T cell immunity. Here, we propose a multifaceted study employing genetic, biochemical, immunological approaches and whole animal experimentation to comprehensively examine the molecular and cellular mechanisms underlying miR-23 cluster-mediated immune regulation. In particular, while we will expand our efforts in studying this miRNA family in T cell immunity with a new focus on CD8+ T cell-mediated immune responses, we will also examine their potential new roles in both Tfh cells and B cells that are crucial for establishment of germinal center (GC) reactions and the resultant humoral immunity. Next, by combining RNA-seq, ChIP-seq approaches with newly developed IR-CLASH technology, we will explore the putative molecular mechanisms underlying miR-23 cluster-dependent immune regulation through identification of genes that are regulated in miR-23 cluster-dependent manner and through identification of targets that are directly controlled by miR-23 cluster. Collective, the over-arching goal of the current proposal is to not only establish a powerful model to dissect the molecular orchestration of cellular differentiation, function, and homeostasis in the adaptive immune system but also build a solid foundation with which to target the miR-23~27~24 family to combat infections and a wide array of human immunological diseases.